Team:Chiba/Notebook 2

From 2010.igem.org

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<html><br><br><br>
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<body bgcolor="white" text="black" link="yellow" vlink="purple" alink="red" background="https://static.igem.org/mediawiki/2010/a/a1/Chiba_ground_2.jpg">
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                <title>Free CSS Navigation Menu Designs 2 at exploding-boy.com</title>
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                        <div id="tabsE">
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                                        <!-- CSS Tabs -->
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<li><a href="https://2010.igem.org/Team:Chiba"><span>Home</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba"><span>Home</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Team"><span>Team</span></a>
+
<li><a href="https://2010.igem.org/Team:Chiba/Team"><span>Team</span></a></li>
-
<ul style="z-index:1"> <br>
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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a></li>
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Team">Team</a></li> <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Member">Member</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/School">School</a></li>
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</ul>
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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a>
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<ul style="z-index:1"><br>
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                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Project">Overall Project</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Circuit_1">Circuit 1</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Circuit_2">Circuit 2</a></li>
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-
</ul>
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-
</li>
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<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Result"><span>Result</span></a>
+
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
-
<ul style="z-index:1"><br>
+
-
                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid1">Plasmid 1</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid2">Plasmid 2</a></li>
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-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/lux_promoter">lux Promoter</a></li><br>
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-
</ul>
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-
</li>
+
-
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a>
+
-
<ul style="z-index:1"><br>
+
-
                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Notebook">Notebook 1</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Notebook_2">Notebook 2</a></li>
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-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Notebook_3">Notebook 3</a></li><br>
+
-
</ul>
+
-
</li>
+
-
 
+
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/Safety"><span>Safety</span></a></li>
+
<li id="current"><a href="https://2010.igem.org/Team:Chiba/Safety"><span>Safety</span></a></li>
 +
 
 +
                        </ul>
 +
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 +
<!--- 1st tab End --->
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                <title>Free CSS Navigation Menu Designs 2 at exploding-boy.com</title>
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                <ul>
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</html>
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                                 <!-- CSS Tabs -->
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<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook 1</span></a></li>
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook_2"><span>Notebook 2</span></a></li>
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook_3"><span>Notebook 3</span></a></li>
 +
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 +
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<br><br><br>
<html><h3>2010-09-21</h3></html>
<html><h3>2010-09-21</h3></html>
PCR
PCR
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  Total               10uL 28uL
  Total               10uL 28uL
  Room temperature 2h
  Room temperature 2h
-
After ligation plasmid is transformed and incubated in LB broth.
+
After ligation plasmid is transformed and incubated in LB broth.
-
cell strain : BL21(DE3), XL10G(each 50uL/tube)
+
cell strain : BL21(DE3), XL10G(each 50uL/tube)
-
plasmid : L1~L4
+
plasmid : L1~L4
   
   
<html><h3>2010-09-24</h3></html>
<html><h3>2010-09-24</h3></html>
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  PstI         1uL               1uL
  PstI         1uL               1uL
  ------------------------------------------------------------------------
  ------------------------------------------------------------------------
-
  total                     50uL                50uL
+
  total         50uL                50uL
  ↓37°C 15min incubator
  ↓37°C 15min incubator
  ↓65°C 20min (sterilization dryer)
  ↓65°C 20min (sterilization dryer)
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   plates of IPTG 0,10,100uM, respectively (total 18 Amp, Cm)
   plates of IPTG 0,10,100uM, respectively (total 18 Amp, Cm)
   ↓37°C
   ↓37°C
-
<br>liquid incubation(Amp, Cm)
+
  liquid incubation(Amp, Cm)
<html><h3>2010-09-29</h3></html>
<html><h3>2010-09-29</h3></html>
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   sequence reaction
   sequence reaction
-
                                       1 2 3 4 5 6
+
                                       1   2   3   4   5   6
   -----------------------------------------------------------------------
   -----------------------------------------------------------------------
   Big dye 3.1 premix                  0.2 0.2 0.2 0.2 0.2 0.2
   Big dye 3.1 premix                  0.2 0.2 0.2 0.2 0.2 0.2
-
   5x sequence buffer                   1 1 1 1 1 1
+
   5x sequence buffer                   1   1   1   1   1   1
-
   plasmid(Ptet-luxR-PT7(ORI)-GFP)       1 1 1
+
   plasmid(Ptet-luxR-PT7(ORI)-GFP)     1   1   1
-
         (Prom-luxR-PT7(ORI)-GFP)               1 1 1
+
         (Prom-luxR-PT7(ORI)-GFP)                 1   1   1
-
   primer(Ptet-luxR-FASTR-rev)           0.5
+
   primer(Ptet-luxR-FASTR-rev)         0.5
-
   5uL  (Ptom-luxR-FASTR-rev)                   0.5
+
   5uL  (Ptom-luxR-FASTR-rev)                     0.5
-
         (PT7/cI-GFP-FASTR-rev)            0.5       0.5
+
         (PT7/cI-GFP-FASTR-rev)            0.5         0.5
-
         (seq-GFP-upstream)                   0.5       0.5
+
         (seq-GFP-upstream)                     0.5         0.5
-
   NFW                               3.3 3.3 3.3 3.3 3.3 3.3
+
   NFW                                 3.3 3.3 3.3 3.3 3.3 3.3
   -----------------------------------------------------------------------
   -----------------------------------------------------------------------
-
   total                               6 6 6 6 6 6
+
   total                               6   6   6   6   6   6
   PCR 96°C(1min)
   PCR 96°C(1min)
   [96°C(15sec)->50°C(5sec)->66°C(2.5min)] x 25cycle
   [96°C(15sec)->50°C(5sec)->66°C(2.5min)] x 25cycle
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   luxR-Ptet-PT7/cI-GFP / luxR-Pc-Plux-cI
   luxR-Ptet-PT7/cI-GFP / luxR-Pc-Plux-cI
     (transformation)
     (transformation)
-
202D(Kan) 2uL
+
  202D(Kan) 2uL
-
XL-10G(Cm) 50uL
+
  XL-10G(Cm) 50uL
-
(double transformation)
+
    (double transformation)
-
2-5 2uL
+
  2-5 2uL
-
202D 2uL
+
  202D 2uL
-
DE3 50uL
+
  DE3 50uL
<html><h3>2010-10-14</h3></html>
<html><h3>2010-10-14</h3></html>
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<html><h3>2010-10-16</h3></html>
<html><h3>2010-10-16</h3></html>
   plasmid1(PCR for Prom-luxR-PT7/cI-GFP)
   plasmid1(PCR for Prom-luxR-PT7/cI-GFP)
-
                                          V I C
+
                                          V   I   C
-
----------------------------------------------------------------------------
+
  ----------------------------------------------------------------------------
-
     template DNA(Ptet-luxR-PT7/cI-GFP)    3 3 3
+
     template DNA(Ptet-luxR-PT7/cI-GFP)    3   3   3
-
     primer  vector fwd   5
+
     primer  vector fwd             5
-
             vector rev(luxR(rev)-Prom(rev) 5
+
             vector rev(luxR(rev)-Prom(rev)5
-
             Ins-fwd(Prom&PT7)             5 5
+
             Ins-fwd(Prom&PT7)                 5   5
-
             Ins-rev(GFP&LVA)               5 5
+
             Ins-rev(GFP&LVA)                 5   5
-
     10x buffer                           5 5  5
+
     10x buffer                             5   5  5
-
     dNTP                               5 5  5
+
     dNTP                                   5   5  5
-
     NFW                               26   26 26
+
     NFW                                   26 26 26
-
     ventP                               1  1 1
+
     ventP                                 1  1   1
-
-----------------------------------------------------------------------------
+
  -----------------------------------------------------------------------------
-
                     total           50  50  50(uL)
+
                     total               50  50  50(uL)
   PCR(HOT start)
   PCR(HOT start)
Line 551: Line 604:
                                                   30cycle
                                                   30cycle
-
V  I C
+
                                          V  I   C
-
----------------------------------------------------------------------------
+
  ----------------------------------------------------------------------------
-
     template DNA(Ptet-luxR-PT7/cI-GFP)    3  3 3
+
     template DNA(Ptet-luxR-PT7/cI-GFP)    3  3   3
-
     primer  vector fwd   3
+
     primer  vector fwd             3
-
             vector rev(luxR(rev)-Prom(rev) 3
+
             vector rev(luxR(rev)-Prom(rev)3
-
             Ins-fwd(Prom&PT7)             3 3
+
             Ins-fwd(Prom&PT7)               3   3
-
             Ins-rev(GFP&LVA)               3 3
+
             Ins-rev(GFP&LVA)                 3   3
-
     10x buffer                            5 5  5
+
     10x buffer                            5   5  5
-
     dNTP                               5 5  5
+
     dNTP                                 5   5  5
-
     NFW                               26   26 26
+
     NFW                                 26 26 26
-
     ventP                                1  1  1
+
     ventP                                1    1  1
-
-----------------------------------------------------------------------------
+
  -----------------------------------------------------------------------------
-
                     total           50  50  50(uL)
+
                     total               50  50  50(uL)
-
PCR(HOT start)
+
  PCR(HOT start)
   94°C 4min –(polymerase 1uL)-> [ 94°C 30sec -> 51°C 30sec -> 72°C 2min ] -> 72°C 10min
   94°C 4min –(polymerase 1uL)-> [ 94°C 30sec -> 51°C 30sec -> 72°C 2min ] -> 72°C 10min
                                                   30cycle
                                                   30cycle
Line 571: Line 624:
   FASTR cloning
   FASTR cloning
   
   
-
----------------------------------------------------------------------
+
  ----------------------------------------------------------------------
-
Vector up                     3           
+
  Vector up                       3           
-
down         3
+
  down         3
-
Insert 1.5        1.5
+
  Insert       1.5        1.5
-
Tango buffer 0.5        0.5
+
  Tango buffer       0.5        0.5
-
T4 Ligase buffer 0.5        0.5
+
  T4 Ligase buffer   0.5        0.5
-
ATP 1         1
+
  ATP                 1         1
-
LguI 0.5         0.5
+
  LguI               0.5       0.5
-
Ligase 0.5         0.5
+
  Ligase             0.5       0.5
-
DpnI 0.5         0.5
+
  DpnI               0.5       0.5
-
NFW 2           2
+
  NFW             2         2
-
-----------------------------------------------------------------------
+
  -----------------------------------------------------------------------
-
Total 10uL 10uL
+
  Total               10uL 10uL
-
Room temperature 2h
+
    Room temperature 2h
Line 595: Line 648:
<html><h3>2010-10-18</h3></html>
<html><h3>2010-10-18</h3></html>
   Insert check colony PCR & liquid incubation
   Insert check colony PCR & liquid incubation
-
Insert Check Colony PCR
+
      Insert Check Colony PCR
                                   up1 up2 up3 down4 down5
                                   up1 up2 up3 down4 down5
-
--------------------------------------------------------------------------
+
--------------------------------------------------------------------------
-
Template(picked colonies)
+
Template(picked colonies)
-
Primer VF 2  2  2    2    2
+
Primer VF         2  2  2    2    2
-
Primer VR 2  2  2    2    2
+
Primer VR         2  2  2    2    2
-
10x Buffer 2  2  2    2    2
+
10x Buffer         2  2  2    2    2
-
dNTP 2  2  2    2    2
+
dNTP                 2  2  2    2    2
-
NFW 10  10  10  10    10  
+
NFW               10  10  10  10    10  
-
Taq DNA Polymerase 1    1    1    1    1
+
Taq DNA Polymerase 1    1    1    1    1
-
--------------------------------------------------------------------------
+
--------------------------------------------------------------------------
-
Total 19  19  19  19    19
+
Total               19  19  19  19    19
-
 
+
   -> PCR  
   -> PCR  
-
94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
+
  94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                   30cycle
                                   30cycle
-
  -> Insert check
+
    -> Insert check
<html><h3>2010-10-19</h3></html>
<html><h3>2010-10-19</h3></html>
Line 623: Line 675:
<html><h3>2010-10-20</h3></html>
<html><h3>2010-10-20</h3></html>
  Insert Check Colony PCR
  Insert Check Colony PCR
-
------------------------------------------------
+
------------------------------------------------
-
Template(picked colonies)
+
Template(picked colonies)
-
Primer VF 2
+
Primer VF             2
-
Primer VR 2
+
Primer VR             2
-
10x Buffer 2
+
10x Buffer             2
-
dNTP 2
+
dNTP                     2
-
NFW 10
+
NFW                   10
-
Taq DNA Polymerase 0.5
+
Taq DNA Polymerase     0.5
-
------------------------------------------------
+
------------------------------------------------
-
Total 18.5
+
Total                   18.5
   PCR  
   PCR  
-
94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
+
  94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                   20cycle
                                   20cycle
<html><h3>2010-10-21</h3></html>
<html><h3>2010-10-21</h3></html>
   It was failed sub-cloning at 20th October. We re-experimented.
   It was failed sub-cloning at 20th October. We re-experimented.
-
Insert plasmid(pSB1A3)
+
Insert plasmid(pSB1A3)
  -----------------------------------
  -----------------------------------
-
DNA 5
+
DNA           5
-
NFW 37.5
+
NFW         37.5
-
NEB Buffer 2 5
+
NEB Buffer 2   5
-
BSA 0.5
+
BSA           0.5
-
EcoRI 1
+
EcoRI           1
-
PstI 1
+
PstI           1
-
----------------------------------
+
----------------------------------
-
total                   50㎕
+
  total         50㎕
-
 
+
    -> gel electrophoresis, ZYMO clean
-
-> gel electrophoresis, ZYMO clean
+
<html><h3>2010-10-22</h3></html>
<html><h3>2010-10-22</h3></html>
   Digestion of pSB3C5
   Digestion of pSB3C5
-
  -----------------------------------
+
  -----------------------------------
-
DNA 4
+
  DNA     4
-
NFW 38.5
+
  NFW           38.5
-
NEB Buffer 2 5
+
  NEB Buffer2      5
-
BSA 0.5
+
  BSA           0.5
-
EcoRI 1
+
  EcoRI           1
-
PstI 1
+
  PstI           1
-
----------------------------------
+
  ----------------------------------
-
total                   50㎕
+
  total           50㎕
<html><h3>2010-10-23</h3></html>
<html><h3>2010-10-23</h3></html>
Line 669: Line 720:
   ----------------------------------------------
   ----------------------------------------------
           insert      3        1
           insert      3        1
-
   pSB3C5 vector     2        1
+
   pSB3C5 vector       2        1
       10x buffer      1        1
       10x buffer      1        1
           ligase      1        1
           ligase      1        1
-
       10x ATP       1        1
+
       10x ATP         1        1
-
           NFW       2        5
+
           NFW         2        5
   ----------------------------------------------
   ----------------------------------------------
           total      10      10  uL
           total      10      10  uL
Line 680: Line 731:
<html><h3>2010-10-24</h3></html>
<html><h3>2010-10-24</h3></html>
  Colony PCR
  Colony PCR
-
------------------------------------------------
+
------------------------------------------------
-
Template(picked colonies)
+
Template(picked colonies)
-
Primer VF 2
+
Primer VF           2
-
Primer VR 2
+
Primer VR           2
-
10x Buffer 2
+
10x Buffer           2
-
dNTP 2
+
dNTP                   2
-
NFW 11.5
+
NFW                   11.5
-
Taq DNA Polymerase 0.5
+
Taq DNA Polymerase 0.5
-
------------------------------------------------
+
------------------------------------------------
-
Total 20uL
+
Total 20uL
  PCR  
  PCR  
-
94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
+
  94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                   20cycle
                                   20cycle
-
   Vector digestion        1    2     3    4    5
+
   Vector digestion        1    2   3    4    5
   ----------------------------------------------------------
   ----------------------------------------------------------
-
DNA 1-3A(pSB1C3) 10 4
+
  DNA 1-3A(pSB1C3)   10                 4
-
J04450(pSB3C5) 10
+
  J04450(pSB3C5)         10
-
2-9A(pSB1A3) 10    4
+
  2-9A(pSB1A3)             10    4
-
NFW 30.5 30.5 30.5 30.5 30.5
+
  NFW                 30.5 30.5 30.5 30.5 30.5
-
NEB Buffer 3 5     5    5    5    5
+
  NEB Buffer 3             5   5    5    5    5
-
BSA 0.5   0.5   0.5  0.5  0.5
+
  BSA                   0.5 0.5 0.5  0.5  0.5
-
EcoRI 2     2    2     2   2
+
  EcoRI             2   2    2   2   2
-
PstI 2     2    2     2   2
+
  PstI             2   2    2   2   2
-
---------------------------------------------------------
+
  ---------------------------------------------------------
-
total                  50     50  50  50 50uL
+
  total                  50   50  50  50   50uL
<html><h3>2010-10-26</h3></html>
<html><h3>2010-10-26</h3></html>
  GFP-LVA didn’t shine. So we did PCR for eliminating LVA from GFP.
  GFP-LVA didn’t shine. So we did PCR for eliminating LVA from GFP.
-
---------------------------------------------------------
+
---------------------------------------------------------
     template DNA Plasmid1 pSB1A3        3   
     template DNA Plasmid1 pSB1A3        3   
                   2-9A(pSB1A3)              3
                   2-9A(pSB1A3)              3
-
     primer  TcIg FA-R   5
+
     primer  TcIg FA-R           5
-
             Pc-luxR FA-R                 5
+
             Pc-luxR FA-R               5
             pSB1C3 FA-R                    5   
             pSB1C3 FA-R                    5   
             pSB1C3 FA-F                    5  
             pSB1C3 FA-F                    5  
-
     10x buffer                           5 5
+
     10x buffer                         5   5
-
     dNTP                                5 5
+
     dNTP                                5   5
-
     NFW                                26 26
+
     NFW                                26 26
-
   ventP                                 1 1
+
   ventP                               1   1
-
---------------------------------------------------------
+
---------------------------------------------------------
-
                     total           50  50 uL
+
                     total             50  50 uL
  PCR  
  PCR  
-
94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
+
  94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                   30cycle
                                   30cycle
   FASTR cloning
   FASTR cloning
-
--------------------------------------------
+
--------------------------------------------
-
Vector                        2   
+
Vector                        2   
-
Insert 4.5       
+
Insert                     4.5       
-
Tango buffer 0.5         
+
Tango buffer               0.5         
-
T4 Ligase buffer 0.5         
+
T4 Ligase buffer       0.5         
-
ATP 1           
+
ATP                         1           
-
LguI 0.5         
+
LguI                       0.5         
-
Ligase 0.5         
+
Ligase                       0.5         
-
DpnI 0.5         
+
DpnI                       0.5         
-
NFW 0           
+
NFW                         0           
-
--------------------------------------------
+
--------------------------------------------
-
Total 10uL
+
Total                       10uL
-
Room temperature 2h
+
  Room temperature 2h

Latest revision as of 19:36, 27 October 2010




 

 




2010-09-21

PCR 

Because it didn’t come out vector PCR(gel electrophoresis), 
we operated re-experiment to change template and template amounts.

Using template
 1. Biobrick 2010 1-3A(pSB1C3)
 2. Biobrick 2010 pSB1C3
 3. Biobrick 2010 2-9A(pSB1A3)
 4. control

Using primer
 Fwd : pSB1C3 FASTR-F
 Rev : pSB1C3 FASTR-R

 -------------------------
 template       5uL
 primer Fwd     5uL
 primer Rev     5uL
 dNTP           5uL
 10xbuffer      5uL
 NFW            24uL
 VentP          1uL
 -------------------------
                50uL

PCR(TAKARA)
 94°C – 5min
 94°C – 15sec    --
 51°C – 30sec      │- 25 cycles
 72°C – 1min     -- 
 72°C – 10min

Vector PCR 

Using template
 1. Biobrick 2010 1-3A(pSB1C3)
 2. Biobrick 2010 pSB1C3
 3. Biobrick 2010 2-9A(pSB1A3)
 4. control

 -------------------------
 template      3uL
 primer Fwd    5uL
 primer Rev    5uL
 dNTP          5uL
 10xbuffer     5uL
 NFW           26uL
 VentP         1uL
 -------------------------
               50uL

Using primer
 Fwd : pSB1C3 FASTR-F
 Rev : pSB1C3 FASTR-R

PCR Condition
 94°C – 5min
 94°C – 30sec      --
 51°C – 30sec        │-25 cycles
 72°C – 2min       --
 72°C – 10min

2010-09-22

Biobrick 2010 2-9A(pSB1A3) -> Gel extraction Gel Electrophoresis 

Biobrick 2010 2-9A(pSB1A3), Biobrick 2010 1-3A(pSB1C3), Biobrick 2010 pSB1C3, Biobrick 2010 2-9A(pSB1A3)
As a result, it didn’t come out band of 1-3A and pSB1C3. 
We concluded re-experiment from liquid incubation to mini-prep. Must check gel electrophoresis band after mini-prep.

FASTR Cloning (Plasmid1) 

Vector (about 2kbp)
-pSB1A3 (conservative solution 100ng/uL)

Insert (about 1kbp)
 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)

master mix
---------------------------------------------------------------------
Vector(50ng)         	0.5	2
Insert(75ng)	          3     -
Tango buffer	        0.5	2
T4 Ligase buffer	0.5	2
ATP	                  1	4
LguI	                0.5	2
Ligase           	0.5	2
DpnI	                0.5	2
NFW	                  3	12
-----------------------------------------------------------------------
Total           	10uL	28uL
Room temperature 2h

Transformation 

Using cell strain : BL21(DE3) 50uL
Using plasmid
 L1 : Pet-LuxR-PT7/cI(OR2)-GFP   5uL
 L2 : Pet-LuxR-PT7/cI(OR1)-GFP   5uL
 L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL
 L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL

After transformation 

We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates.
※A Reason of using DE3 cell strain
DE3 cell strain has T7 RNA Polymerase gene in genome, and under IPTG derivation T7RNAP is expressed.
Plasmid1 has  T7/cI hybrid promoter. 
So if this promoter is accelerated by T7RNAP, GFP is expressed and shines green. 
In other words, we used DE3 cell strain to check T7 accelerating function of PT7/cI.

2010-09-23

From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3. And then it is eluted by 50uL Elution Solution. ↓ Gel Electrophoresis(1uL) 

L1 : Pet-LuxR-PT7/cI(OR2)-GFP   5uL
L2 : Pet-LuxR-PT7/cI(OR1)-GFP   5uL
L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5uL
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5uL
Remaining of above sample is transformed and incubated.
cell strain : XL10G(50uL/tube)
plasmid : L1~L4(total 4)

FASTR Cloning (Plasmid1) 

Vector (about 2kbp)
 -pSB1A3 (conservative solution 100ng/uL)
Insert (about 1kbp)
 1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
 3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/uL)
 4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/uL)
master mix
---------------------------------------------------------------------
Vector(50ng)         	0.5	2
Insert(75ng)	          3	-
Tango buffer	        0.5	2
T4 Ligase buffer	0.5	2
ATP	                  1	4
LguI	                0.5	2
Ligase	                0.5	2
DpnI	                0.5	2
NFW	                  3	12
-----------------------------------------------------------------------
Total	               10uL	28uL
Room temperature 2h
After ligation plasmid is transformed and incubated in LB broth.
cell strain : BL21(DE3), XL10G(each 50uL/tube)
plasmid : L1~L4

2010-09-24

At 23th September Remaining of above sample is transformed and incubated. cell strain : XL10G(50uL/tube) plasmid : L1~L4(total 4) We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.
Insert Check Colony PCR(Plasmid 1) 

master mix
--------------------------------------------------------------------------
Template(picked colonies)
Primer VF	         2uL	40uL
Primer VR	         2uL	40uL
10x Buffer	         2uL	40uL
dNTP	                 2uL	40uL
NFW	                10uL	200uL
Taq DNA Polymerase	0.5uL	5uL
--------------------------------------------------------------------------
Total	                20uL	400uL
↓
PCR
 94°C – 5min
 94°C – 30sec      --
 51°C – 30sec        │-20 cycles
 72°C – 1min       --
 72°C – 10min
↓
Gel Electrophoresis

2010-09-25

Mini-prep and gel electrophoresis of liquid culture medium at 24th September. 

Gel Electrophoresis picture
↓
Transformation to BL21(DE3) 
↓
Incubation in LB broth, IPTG(0, 10, 100 μM) 
↓37°C
About total sample, it is shown GFP ON/OFF switching on eyes.
↓
For the purpose of measuring fluorescence level, we did liquid incubation from each plate.
IPTG(0, 10, 100 μM) x 6 samples = 18 test tubes
↓37°C
∙ measuring fluorescence level(liquid incubation)
∙ pelletization of cell

Digestion and ligation of BBa-J01011(Ptet-cI) and pSB3C5 

(Digestion)
           Insert(J01011)	   Vector(pSB3C5)
DNA	       10uL	                10uL
NFW	       32.5uL	               32.5uL
NEB Buffer 2	5uL	                5uL
BSA	       0.5uL	               0.5uL
EcoRI	        1uL	               1uL
PstI	        1uL	               1uL
------------------------------------------------------------------------
total         50uL                50uL
↓37°C 15min incubator
↓65°C 20min (sterilization dryer)
※After digestion, we did gel electrophoresis

(Ligation)
  13uL NFW
   2uL Insert(digested)
   2uL Vector(digested)                  total 20uL in 0.2mL PCR tube
   2uL 10x T4 DNA Ligase Buffer
   1uL T4 DNA Ligase

↓10min at room temperature
↓20min at 65°C (sterilization dryer)

(Transformation)
Transformation XL10G 50uL to 10uL of ligation product
↓37°C
Insert check of living colonies(colony PCR)     /     liquid incubation
↓total 7 colonies                               ↓37°C
Insert Check Colony PCR(Plasmid 1)	                    ↓We did mini-prep only No.4 sample checked insert.
--------------------------------------------
Template(picked colonies)                                  ↓
Primer VF	2uL	                                    Gel electrophoresis
Primer VR	2uL	
10x Buffer	2uL
dNTP	2uL
NFW	11.5uL
Taq DNA Polymerase	0.5uL
--------------------------------------------------------
Total	20uL
↓
PCR
 94°C 5min
 94°C 30sec   --
 51°C 30sec     │-30 cycle
 72°C 1min    --
 72°C 10min
↓
Gel electrophoresis

2010-09-27

 double transformation
  DE3 50uL + Ptet-cI 2uL + 1-1/1-2/1-3/2-5/3-3/4-8 2uL
 ↓
 plates of IPTG 0,10,100uM, respectively (total 18 Amp, Cm)
 ↓37°C
 liquid incubation(Amp, Cm)

2010-09-29

 We did gel electrophoresis to check Plux/cII
 Experiment of checking T7/cI promoter fuction

2010-09-30

 pSB1AK3(K228822)
   ↓
 transformation
   ↓
 liquid incubation
   ↓
 spin down
   ↓
 mini-prep

2010-10-02

 Checking plasmid(gel electrophoresis)
   T7RNAP -> 2-2F(K145001)
   Ptet-cI -> 1-11H(J01101)
   GFP -> 3-12M(K081012)
 transformation(pcI434)
   ↓
 liquid incubation

 Checking cI-LVA
   (1) double transformation of J01101(Ptet-cI) and PcI-GFP(Ryuji) (Amp, Cm)
   (2) transformation PcI-GFP
 cI434-T-T (P0152, pSB1A2, 1-10E) -> 1uL DNA -> transformation to 30uL XL10G(Kan)
 -> liquid incubation

2010-10-03

 Experiment of checking T7/cI promoter
  <plasmid>
    2+ : Ptet-luxR-PT7/cI-GFP(pUC, Amp) + Ptet-cI(pSB3C5)
    2- : Ptet-luxR-PT7/cI-GFP(pUC, Amp)
    4+ : Prom-luxR-PT7/cI-GFP(pUC, Amp) + Ptet-cI(pSB3C5)
    4- : Prom-luxR-PT7/cI-GFP(pUC, Amp)
 strain : BL21(DE3)

 Checking CI-LVA
   Ptet-cI(pAC) / Ptet-cI(pUC, pSB1A2) / PcI-GFP(Ryuji) / PcI-GFP(S03325, pSB1A2)
   strain : XL-10G 50uL
     -> transformation -> incubating in plates

2010-10-04

 continuing 2th October…
  -> spinning down P0152 and R0051 -> mini-prep

 R0052 cI434 promoter
  2010 kit plate2 11-D pSB2K3
   XL-10G(Cm) 50uL
   DNA 2uL
   SOC 200uL
     -> transformation -> gel electrophoresis

2010-10-05

 Mini-prep of R0051 and R0052 was completed.

2010-10-06

 Checking T7/cI promoter function(being stimulated by T7RNAP)
  Prom-luxR-PT7/cI(ORI)-GFP (Amp)
  Pc-T7RNAP
  strain : XL-10G
       -> transformation -> incubation

 Checking luxR of plasmid1
  Plasmid : Ptet-luxR-PT7/cI(ORI)-GFP
           Prom-luxR-PT7/cI(ORI)-GFP
          Plux-GFP
  strain : XL-10G
        -> transformation -> incubation -> liquid incubation -> mini-prep -> gel electrophoresis

2010-10-07

 Checking cI
   Ptet-cI(pAC) / Ptet-cI(pUC) / PcI-GFP(pUC) / PcI-GFP(pAC)
    strain : XL-10G(50uL)
         -> transformation -> incubation

 sequence reaction
                                      1   2   3   4   5   6
 -----------------------------------------------------------------------
 Big dye 3.1 premix                   0.2 0.2 0.2 0.2 0.2 0.2
 5x sequence buffer                   1   1   1   1   1   1
 plasmid(Ptet-luxR-PT7(ORI)-GFP)      1   1   1
        (Prom-luxR-PT7(ORI)-GFP)                  1   1   1
 primer(Ptet-luxR-FASTR-rev)          0.5
  5uL  (Ptom-luxR-FASTR-rev)                      0.5
       (PT7/cI-GFP-FASTR-rev)             0.5         0.5
       (seq-GFP-upstream)                     0.5         0.5
 NFW                                  3.3 3.3 3.3 3.3 3.3 3.3
 -----------------------------------------------------------------------
  total                               6   6   6   6   6   6
 PCR 96°C(1min)
  [96°C(15sec)->50°C(5sec)->66°C(2.5min)] x 25cycle
     -> 8°C hold -> 75% isopropyl -> centrifuge(10min, max) -> 75% isopropyl -> centrifuge
     -> drying at 65°C -> heatshock(95°C, 3min)

2010-10-13

 luxR-Ptet-PT7/cI-GFP / luxR-Pc-Plux-cI
   (transformation)
 202D(Kan) 2uL
 XL-10G(Cm) 50uL

   (double transformation)
 2-5 2uL
 202D 2uL
 DE3 50uL

2010-10-14

 liquid incubation 202D, 202D/2-5
 checking function of PT7/cI

2010-10-16

 plasmid1(PCR for Prom-luxR-PT7/cI-GFP)
                                          V   I   C
 ----------------------------------------------------------------------------
   template DNA(Ptet-luxR-PT7/cI-GFP)     3   3   3
   primer   vector fwd    	           5
            vector rev(luxR(rev)-Prom(rev)5
            Ins-fwd(Prom&PT7)                 5   5
            Ins-rev(GFP&LVA)                  5   5
   10x buffer                             5   5   5
   dNTP                                   5   5   5
   NFW                                   26  26  26
   ventP                                  1   1   1
 -----------------------------------------------------------------------------
                    total                50  50  50(uL)

 PCR(HOT start)
  94°C 4min –polymerase 1uL-> [ 94°C 30sec -> 51°C 30sec -> 72°C 2min ] -> 72°C 10min
                                                 30cycle

                                          V  I   C
 ----------------------------------------------------------------------------
   template DNA(Ptet-luxR-PT7/cI-GFP)     3  3   3
   primer   vector fwd    	           3
            vector rev(luxR(rev)-Prom(rev)3
            Ins-fwd(Prom&PT7)                3   3
            Ins-rev(GFP&LVA)                 3   3
   10x buffer                            5   5   5
   dNTP                                  5   5   5
   NFW                                  26  26  26
   ventP                                1    1   1
 -----------------------------------------------------------------------------
                    total               50  50  50(uL)

 PCR(HOT start)
  94°C 4min –(polymerase 1uL)-> [ 94°C 30sec -> 51°C 30sec -> 72°C 2min ] -> 72°C 10min
                                                 30cycle

 FASTR cloning

 ----------------------------------------------------------------------
 Vector up                       3          
 down          	3
 Insert	      1.5        1.5
 Tango buffer	      0.5        0.5
 T4 Ligase buffer    0.5        0.5
 ATP	                1         1
 LguI	              0.5        0.5
 Ligase              0.5        0.5
 DpnI	              0.5        0.5
 NFW             	2         2
 -----------------------------------------------------------------------
 Total	              10uL	10uL
   Room temperature 2h


2010-10-17

 Experiment of checking function PT7/cI
  (1) Plux-cI(LVA) RBS : weak
                                  + PT7/cI-GFP
  (2) Plux-cI(LVA) RBS : strong

2010-10-18

 Insert check colony PCR & liquid incubation
      Insert Check Colony PCR
                                 up1 up2 up3 down4 down5
--------------------------------------------------------------------------
Template(picked colonies)
Primer VF	        2   2   2     2     2
Primer VR	        2   2   2     2     2
10x Buffer	        2   2   2     2     2
dNTP	                2   2   2     2     2
NFW	               10   10   10   10    10 
Taq DNA Polymerase	1    1    1    1     1
--------------------------------------------------------------------------
Total	               19   19   19   19    19
 -> PCR 
  94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                  30cycle
    -> Insert check

2010-10-19

 Cloning of plasmid1(Prom-luxR-PT7/cI-GFP) -> pSB1C3(for submission), pSB3C5(for experiment)
 
 Pre-culture for checking Prom-luxR function
  Prom-luxR-PT7/cI-GFP-pSB1A3(pUC)  (Amp)
  Plux-GFP(pAC)  (Cm)
       -> liquid incubation -> measuring OD and fluorescence degree

2010-10-20

Insert Check Colony PCR
------------------------------------------------
Template(picked colonies)
Primer VF	            2
Primer VR	            2
10x Buffer	            2
dNTP	                    2
NFW	                   10
Taq DNA Polymerase	    0.5
------------------------------------------------
Total	                   18.5

 PCR 
  94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                  20cycle

2010-10-21

 It was failed sub-cloning at 20th October. We re-experimented.
Insert plasmid(pSB1A3)
-----------------------------------
DNA	          5
NFW	         37.5
NEB Buffer 2	  5
BSA	          0.5
EcoRI            1
PstI	          1
----------------------------------
 total          50㎕
    -> gel electrophoresis, ZYMO clean

2010-10-22

 Digestion of pSB3C5
 -----------------------------------
 DNA    	   4
 NFW	          38.5
 NEB Buffer2      5
 BSA	           0.5
 EcoRI	           1
 PstI 	           1
 ----------------------------------
 total            50㎕

2010-10-23

 Ligation reaction        positive control
 ----------------------------------------------
         insert      3        1
 pSB3C5 vector       2        1
     10x buffer      1        1
         ligase      1        1
     10x ATP         1        1
         NFW         2        5
 ----------------------------------------------
         total       10       10   uL
 Room temperature(2h)

2010-10-24

Colony PCR
------------------------------------------------
Template(picked colonies)
Primer VF	          2
Primer VR	          2
10x Buffer	          2
dNTP	                  2
NFW                   11.5
Taq DNA Polymerase	0.5
------------------------------------------------
Total	20uL

PCR 
 94°C 2min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                  20cycle

  Vector digestion         1    2    3    4    5
  ----------------------------------------------------------
  DNA 1-3A(pSB1C3)	   10	                4
  J04450(pSB3C5)	        10
  2-9A(pSB1A3)		             10    4
  NFW	                 30.5 30.5 30.5 30.5 30.5
  NEB Buffer 3	            5    5    5    5    5
  BSA	                  0.5  0.5  0.5  0.5  0.5
  EcoRI	            2    2    2    2    2
  PstI 	            2    2    2    2    2
  ---------------------------------------------------------
  total                   50   50   50   50   50uL

2010-10-26

GFP-LVA didn’t shine. So we did PCR for eliminating LVA from GFP.
---------------------------------------------------------
   template DNA Plasmid1 pSB1A3        3  
                 2-9A(pSB1A3)              3
   primer   TcIg FA-R    	        5
            Pc-luxR FA-R               5
            pSB1C3 FA-R                    5   
            pSB1C3 FA-F                    5 
   10x buffer                          5   5
   dNTP                                5   5
   NFW                                26  26
  ventP                                1   1
---------------------------------------------------------
                    total             50  50 uL

PCR 
  94°C 5min --> [ 94°C 30sec -> 50°C 30sec -> 72°C 2min ] -> 72°C 10min
                                  30cycle

 FASTR cloning
--------------------------------------------
Vector                         2   
Insert        	              4.5       
Tango buffer	              0.5        
T4 Ligase buffer	      0.5        
ATP	                        1          
LguI	                      0.5        
Ligase	                      0.5        
DpnI	                      0.5        
NFW	                        0          
--------------------------------------------
Total	                       10uL
 Room temperature 2h
Retrieved from "http://2010.igem.org/Team:Chiba/Notebook_2"